U.S. patent application number 12/637833 was filed with the patent office on 2010-07-01 for anti-idiotype antibody against an antibody against the amyloid beta peptide.
Invention is credited to ULRICH ESSIG, WOLFGANG HOESEL, KAY-GUNNAR STUBENRAUCH, RUDOLF VOGEL.
Application Number | 20100167313 12/637833 |
Document ID | / |
Family ID | 40581970 |
Filed Date | 2010-07-01 |
United States Patent
Application |
20100167313 |
Kind Code |
A1 |
ESSIG; ULRICH ; et
al. |
July 1, 2010 |
ANTI-IDIOTYPE ANTIBODY AGAINST AN ANTIBODY AGAINST THE AMYLOID BETA
PEPTIDE
Abstract
The present invention is directed to an anti-idiotype antibody
binding to the complementary determining region of an antibody
against the amyloid .beta. peptide. In one embodiment said antibody
binds to the same epitope or an overlapping epitope as the antibody
obtainable from the cell line DSM ACC2939. Also reported is an
immunoassay for the determination of an antibody against the
amyloid .beta. peptide and for determination of an anti-idiotype
antibody binding to an antibody against the amyloid .beta.
peptide.
Inventors: |
ESSIG; ULRICH; (PLANEGG,
DE) ; HOESEL; WOLFGANG; (TUTZING, DE) ;
STUBENRAUCH; KAY-GUNNAR; (PENZBERG, DE) ; VOGEL;
RUDOLF; (WEILHEIM, DE) |
Correspondence
Address: |
HOFFMANN-LA ROCHE INC.;PATENT LAW DEPARTMENT
340 KINGSLAND STREET
NUTLEY
NJ
07110
US
|
Family ID: |
40581970 |
Appl. No.: |
12/637833 |
Filed: |
December 15, 2009 |
Current U.S.
Class: |
435/7.9 ;
530/387.2 |
Current CPC
Class: |
C07K 16/4241 20130101;
G01N 33/686 20130101; C07K 2317/565 20130101; G01N 2469/20
20130101; C07K 2317/56 20130101; G01N 2333/4709 20130101 |
Class at
Publication: |
435/7.9 ;
530/387.2 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 16/42 20060101 C07K016/42 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2008 |
EP |
08022235.9 |
Claims
1. An antibody that binds to a complementary determining region of
an antibody against the amyloid .beta. peptide and binds to the
same epitope, or an overlapping epitope, as the antibody obtained
from the cell line DSM ACC2939.
2. The antibody of claim 1, wherein the heavy chain variable domain
of the antibody against the amyloid .beta. peptide comprises a CDR3
with an amino acid sequence selected from SEQ ID NO: 1, 2, and 3
and the light chain variable domain of said antibody against the
amyloid .beta. peptide comprises a CDR3 with an amino acid sequence
selected from SEQ ID NO: 4, 5, and 6.
3. An antibody derived from the cell line DSM ACC2939.
4. A method for the immunological determination of an antibody
against the amyloid .beta. peptide in a sample comprising an
immunoassay employing an antibody obtained from cell line DSM
ACC2939.
5. The method of claim 4, wherein the immunoassay comprises a
capture antibody, a tracer antibody and a detection antibody,
wherein said capture antibody is a biotinylated anti-idiotype
antibody against an antibody against the amyloid .beta. peptide
obtained from cell line DSM ACC2939 conjugated to a solid phase via
streptavidin, said tracer antibody is an anti-idiotype antibody
against an antibody against the amyloid .beta. peptide obtained
from cell line DSM ACC2939 conjugated to digoxygenin as detectable
label, and said detection antibody is an antibody against
digoxygenin conjugated to a peroxidase.
6. The method of claim 4, wherein the immunoassay comprises the
amyloid .beta. peptide conjugated to a solid phase, an
anti-idiotype antibody against an antibody against the amyloid
.beta. peptide obtained from cell line DSM ACC2939 conjugated to
digoxygenin as detectable label, and a detection antibody against
digoxygenin conjugated to a peroxidase.
7. The method of claim 4, wherein the immunoassay comprises a
capture antibody and a tracer antibody, wherein said capture
antibody is a mixture comprising at least two of said antibodies
that differ in the antibody site at which they are conjugated to
the solid phase, and the tracer antibody is a mixture of said
antibody comprising at least two of said antibodies that differ in
the antibody site at which they are conjugated to the detectable
label.
8. A method for the immunological determination of an anti-idiotype
antibody against an antibody against the amyloid .beta. peptide in
a sample comprising an immunoassay employing an antibody obtained
from cell line DSM ACC2939.
9. The method of claim 8, wherein the immunoassay comprises a
capture antibody and a tracer antibody, wherein said capture
antibody is an antibody against the amyloid .beta. peptide
conjugated to a first part of a binding pair and said tracer
antibody is an antibody against the amyloid .beta. peptide
conjugated to a detectable label.
10. The method of claim 8, wherein the immunoassay comprises the
amyloid .beta. peptide conjugated to a solid phase, an antibody
against the amyloid .beta. peptide conjugated to digoxygenin as
detectable label, and a detection antibody against digoxygenin
conjugated to a peroxidase.
11. The method of claim 8, wherein the immunoassay comprises a
capture antibody and a tracer antibody, wherein said capture
antibody is a mixture comprising at least two of said antibodies
that differ in the antibody site at which they are conjugated to
the solid phase, and the tracer antibody is a mixture of said
antibody comprising at least two of said antibodies that differ in
the antibody site at which they are conjugated to the detectable
label.
Description
PRIORITY TO RELATED APPLICATION(S)
[0001] This application claims the benefit of European Patent
Application No. 08022235.9, filed Dec. 22, 2008, which is hereby
incorporated by reference in its entirety.
[0002] The current invention is directed to an anti-idiotype
antibody binding to an anti-A.beta. antibody and an assay for
detecting antibodies binding to the same or an overlapping epitope
on an anti-A.beta. antibody.
BACKGROUND OF THE INVENTION
[0003] About 70% of all cases of dementia are due to Alzheimer's
disease which is associated with the selective damage of brain
regions and neural circuits critical for cognition Alzheimer's
disease is characterized by neurofibrillary tangles in particular
in pyramidal neurons of the hippocampus and numerous amyloid
plaques containing mostly a dense core of amyloid deposits and
defused halos.
[0004] The extracellular neuritic plaques contain large amounts of
a pre-dominantly fibrillar peptide termed "amyloid .beta.",
"A-beta", "A.beta.4", ".beta.-A4" or "A.beta." (see e.g. Selkoe, D.
J., Ann. Rev. Cell Biol. 10 (1994) 373-403; Koo, E. H., Proc. Natl.
Acad. Sci. USA 96 (1999) 9989-9990; U.S. Pat. No. 4,666,829; or
Glenner, G. G., Biochem. Biophysic. Res. Commun. 122 (1984)
1131-1135). This amyloid .beta. peptide is derived from "Alzheimer
precursor protein/.beta.-amyloid precursor protein" (APP). APPs are
integral membrane glycoproteins (see e.g. Sisodia, S. S., Proc.
Natl. Acad. Sci. USA 89 (1992) 6075-6079) and are
endoproteolytically cleaved within the A.beta. sequence by a plasma
membrane protease, .alpha.-secretase (see e.g. Sisodia (1992), loc.
cit.). Furthermore, further secretase activity, in particular
.beta.-secretase and .gamma.-secretase activity, leads to the
extracellular release of amyloid-.beta.(A.beta.) comprising either
39 amino acids (A.beta.39), 40 amino acids (A.beta.40), 42 amino
acids (A.beta.42) or 43 amino acids (A.beta.43) (see e.g. Sinha,
S., Proc. Natl. Acad. Sci. USA 96 (1999) 11049-11053; Price, D. L.,
Science 282 (1998) 1079-1083; WO 00/72880; or Hardy, J., Trends in
Neuroscience (1997) 154-159).
[0005] It is of note that A.beta. has several natural occurring
forms, whereby the human forms are referred to as the above
mentioned A.beta.39, A.beta.40, A.beta.41, A.beta.42 and A.beta.43.
The most prominent form, A.beta.42, has the amino acid sequence
(starting from the N-terminus):
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 13). In
A.beta.41, A.beta.40, A.beta.39, the C-terminal A, IA and VIA,
respectively, are missing. In the A.beta.43-form an additional
threonine residue is comprised at the C-terminus of the above
depicted sequence.
[0006] Standard solid-phase immunoassays with antibodies involve
the formation of a complex between an antibody adsorbed/immobilized
on a solid phase (capture antibody), the antigen, and an antibody
to another epitope of the antigen conjugated with an enzyme or
detectable label (tracer antibody). In the assay, a sandwich is
formed: solid phase/capture antibody/antigen/tracer antibody. In
the reaction catalyzed by the sandwich among other things the
activity of the antibody-conjugated enzyme is proportional to the
antigen concentration in the incubation medium. The standard
sandwich method is also called double antigen bridging immunoassay
because capture and tracer antibodies bind to different epitopes of
the same antigen. Hoesel, W., et al. (J. Immunol. Methods 294
(2004) 101-110) report an anti-EPO double antigen bridging assay
wherein a mixture of immobilized rhEPO coupled to amino groups and
to carbohydrate groups was used. Immunoassays such as the double
antigen bridging ELISA are common assay types in the investigation
of an immunogenic answer of a patient to a drug antibody.
Mire-Sluis, A. R., et al. (J. Immunol. Methods 289 (2004) 1-16)
summarize the recommendations for the design and optimization of
immunoassays using detection of host antibodies against
biotechnology products. Anti-drug antibody assays are mentioned,
for example, in WO 2005/045058 and WO 90/006515. Anti-idiotypic
antibody assays are mentioned, for example, in U.S. Pat. No.
5,219,730; WO 87/002778; EP 0 139 389; and EP 0 170 302. Wadhwa,
M., et al. (J. Immunol. Methods 278 (2003) 1-17) report strategies
for the detection, measurement and characterization of unwanted
antibodies induced by therapeutic biologicals. In US 2007/0093415
amyloid specific peptides and uses thereof are reported. A method
for producing anti idiotypic antibodies is reported in EP 1 917
854.
SUMMARY OF THE INVENTION
[0007] The first aspect of the current invention is an
anti-idiotype antibody binding to an antibody against the amyloid
.beta. peptide as well as its use in an assay according to the
invention. In one embodiment said antibody is characterized in
binding to the same or an overlapping epitope as the antibody that
is obtained from the deposited cell line DSM ACC2939. Another
embodiment is that said antibody is the antibody that is obtained
from the deposited cell line DSM ACC2939.
[0008] Other aspects of the invention are methods for the
immunological determination of an antibody against the amyloid
.beta. peptide in a sample using an immunoassay and for the
immunological determination of an anti-idiotype antibody against an
antibody against the amyloid .beta. peptide in a sample using an
immunoassay.
[0009] In an embodiment of the method for the immunological
determination of an antibody against the amyloid .beta. peptide in
a sample the immunoassay comprises a capture antibody, a tracer
antibody and a detection antibody, wherein the capture antibody is
a biotinylated anti-idiotype antibody against an antibody against
the amyloid .beta. peptide conjugated to a solid phase via
streptavidin, the tracer antibody is an anti-idiotype antibody
against an antibody against the amyloid .beta. peptide conjugated
to digoxygenin as detectable label, and the detection antibody is
an antibody against digoxygenin conjugated to horseradish
peroxidase.
[0010] In a further embodiment of the method for the immunological
determination of an antibody against the amyloid .beta. peptide in
a sample the immunoassay comprises the amyloid .beta. peptide
conjugated to a solid phase, an anti-idiotype antibody against an
antibody against the amyloid .beta. peptide conjugated to
digoxygenin as detectable label, and a detection antibody against
digoxygenin conjugated to horseradish peroxidase.
[0011] In one embodiment the anti-idiotype antibody against an
antibody against the amyloid .beta. peptide binds to the same or an
overlapping epitope as the antibody obtained from the deposited
cell line DSM ACC2939. In another embodiment the anti-idiotype
antibody against an antibody against the amyloid .beta. peptide is
the antibody obtained from the deposited cell line DSM ACC2939.
[0012] In one embodiment of the method for the immunological
determination of an anti-idiotype antibody against an antibody
against the amyloid .beta. peptide in a sample the immunoassay
comprises a capture antibody and a tracer antibody, wherein the
capture antibody is an antibody against the amyloid .beta. peptide
conjugated to a first part of a binding pair and the tracer
antibody is an antibody against the amyloid .beta. peptide
conjugated to a detectable label. In one embodiment comprises the
immunoassay a detection antibody against digoxygenin conjugated to
horseradish peroxidase.
[0013] In a further embodiment of the method for the immunological
determination of an anti-idiotype antibody against an antibody
against the amyloid .beta. peptide in a sample the immunoassay
comprises the amyloid .beta. peptide conjugated to a solid phase,
an antibody against the amyloid .beta. peptide conjugated to
digoxygenin as detectable label, and a detection antibody against
digoxygenin conjugated to horseradish peroxidase.
[0014] In one embodiment of the method according to the invention
the immunoassay comprises a capture antibody and a tracer antibody,
wherein the capture antibody is a mixture comprising at least two
antibodies that differ in the antibody site at which they are
conjugated to the solid phase, and the tracer antibody is a mixture
comprising at least two antibodies that differ in the antibody site
at which they are conjugated to the detectable label.
[0015] In one embodiment the conjugation of the antibody to its
conjugation partner is performed by chemically binding via
N-terminal and/or .epsilon.-amino groups (lysine), .epsilon.-amino
groups of different lysines, carboxy-, sulfhydryl-, hydroxyl-
and/or phenolic functional groups of the amino acid backbone of the
drug antibody and/or sugar alcohol groups of the carbohydrate
structure of the drug antibody. In another embodiment the capture
antibody mixture comprises the antibody conjugated via an amino
group and via a carbohydrate structure to its conjugation
partner.
[0016] In one embodiment the antibody against the amyloid .beta.
peptide is the antibody as reported in WO 03/070760. In another
embodiment the antibody against the amyloid .beta. peptide
comprises a heavy chain CDR3 selected from SEQ ID NO: 1, 2 and 3,
and a light chain CDR3 selected from SEQ ID NO: 4, 5 and 6. In a
further embodiment comprises the antibody against the amyloid
.beta. peptide a heavy chain variable domain selected from SEQ ID
NO: 7, 8 and 9 and/or a light chain variable domain selected from
SEQ ID NO: 10, 11 and 12.
[0017] In a further embodiment the capture antibody mixture and/or
the tracer antibody mixture comprise the antibody conjugated via at
least two different amino groups to its conjugation partner. Such
coupling via different amino groups can be performed by acylation
of a part of the .epsilon.-amino groups with chemical protecting
agents, e.g. by citraconylation, in a first step. In a second step
conjugation is performed via the remaining amino groups.
Subsequently citraconylation is removed and the antibody is
conjugated to the conjugation partner via remaining free amino
groups, i.e. the antibody obtained is conjugated to the conjugation
partner via amino groups that have not been protected by
citraconylation. Suitable chemical protecting agents form bonds at
unprotected side chain amines and are less stable than and
different from those bonds at the N-terminus. Many such chemical
protecting agents are known (see for example EP 0 651 761). In one
embodiment the chemical protecting agents include cyclic
dicarboxylic acid anhydrides like maleic or citraconylic acid
anhydride.
[0018] In one embodiment the capture antibody is conjugated to the
solid phase by passive adsorption and therefore is conjugated to
the solid phase at least two different antibody sites. Passive
adsorption is, e.g., described by Butler, J. E., in "Solid Phases
in Immunoassay" (1996) 205-225 and Diamandis, E. P., and
Christopoulos, T. K. (Editors), in "Immunoassays" (1996) Academic
Press (San Diego).
[0019] In one embodiment the tracer antibody mixture comprises the
antibody conjugated via an amino group and via a carbohydrate
structure to its conjugation partner.
[0020] In another embodiment the ratio of capture antibody to
tracer antibody is 1:10 to 50:1 (ratio means molar ratio of the
antibody molecules irrespective of the molecular weight of the
conjugates, which can be different). In still a further embodiment
the ratio of amino conjugated antibody (either tracer or capture
antibody) to carbohydrate conjugated antibody (either tracer or
capture antibody) in such a mixture is 1:10 to 10:1 (ratio means
molar ratio of antibody molecules irrespective of the molecular
weight of the conjugates, which can be different).
[0021] In one embodiment of the invention, the capture antibody is
conjugated (immobilized) via a specific binding pair. Such a
binding pair (first component/second component) is in one
embodiment selected from streptavidin or avidin/biotin,
antibody/antigen (see, for example, Hermanson, G. T., et al.,
Bioconjugate Techniques, Academic Press, 1996),
lectin/polysaccharide, steroid/steroid binding protein,
hormone/hormone receptor, enzyme/substrate, IgG/Protein A and/or G,
etc. In one embodiment the capture antibody is conjugated to biotin
and immobilization is performed via immobilized avidin or
streptavidin.
[0022] In another embodiment the tracer antibody is conjugated to a
detectable label. In one embodiment the tracer antibody is
conjugated via digoxygenin and an antibody against digoxygenin to
the detectable label. Alternatively the tracer antibody is
conjugated to an electrochemiluminescent label, like a ruthenium
bispyridyl complex.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The term "antibody against the amyloid .beta. peptide"
according to the invention denotes an antibody which can be
administered to an individual, so that a sample of said individual
is suspected to comprise said antibody after administration. Within
one assay according to the invention, the capture antibody and the
tracer antibody comprise the "same" antibody molecule, e.g.
recombinantly produced with the same expression vector and
comprising the same amino acid sequence. Antibody against the
amyloid .beta. peptide are described, for example, in U.S. Pat. No.
7,256,273, U.S. Pat. No. 7,189,819, U.S. Pat. No. 7,179,892, U.S.
Pat. No. 7,195,761, US 2008/0281082, US 2008/0221306, US
2008/0131422, US 2008/0050367, US 2007/0238154, US 2007/0154480, US
2007/0110750, US 2006/0280743, US 2006/0292152, US 2006/0165682, US
2006/0057701, US 2006/0057702, US 2006/0039906, US 2005/0249725, US
2005/0169925, US 2005/0118651, US 2005/0009150, US 2004/0171816, US
2004/0171815, and US 2004/0192898.
[0024] "Anti-idiotype antibodies" are antibodies, which are
directed against the antigen binding site, i.e. the variable
region, of a therapeutic antibody, like the complementary
determining region. Such anti-idiotype antibodies may occur during
antibody therapy as an immunogenic reaction of a patient (see e.g.
Pan, Y., et al., FASEB J. 9 (1995) 43-49). In one embodiment said
anti-idiotype antibody against an antibody against the amyloid
.beta. peptide is binding to one or more of the CDR of the antibody
against the amyloid .beta. peptide.
[0025] The first aspect of the current invention is an
anti-idiotype antibody binding to an antibody against the amyloid
.beta. peptide. An exemplary antibody for this aspect of the
invention is the antibody that is obtained from the deposited cell
line DSM ACC2939. This antibody and its use in an assay according
to the invention are also aspects of the current invention. In one
embodiment said anti-idiotype antibody is characterized in binding
to the same or an overlapping epitope as the antibody that is
obtained from the deposited cell line DSM ACC2939. Two epitopes are
overlapping if a signal reduction of 50% or more, in one embodiment
of 75% or more, is detected by a surface plasmon resonance (SPR)
assay using the immobilized antibody and soluble antigen, or vice
versa, with the epitope in question at a concentration of 20-50 nM
and the antibody for which the epitope overlap has to be detected
at a concentration of 100 nM. Alternatively a method can be used in
which epitope overlap of two antibodies binding to the same antigen
is determined with the help of a competitive test system. For this
purpose, for example with the help of a cell-based enzyme
immunoassay (ELISA) employing cells expressing recombinant antigen
epitopes, it is tested if the antibody for which the epitope
overlap has to be detected competes with the other antibody for the
binding to the immobilized antigen. For this purpose, the
immobilized antigen is incubated with the antibody in labeled form
and an excess of the antibody for which the epitope overlap has to
be determined. By detection of the bound labeling there can easily
be ascertained the epitope overlap. If a signal reduction of more
than 70%, in one embodiment of more than 80%, at the same
concentration, or a displacement of more than 80%, in one
embodiment of more than 90%, at higher concentrations, in one case
with a 10.sup.5-fold excess of the antibody for which epitope
overlap has to be determined, referred to the known antibody is
determined then epitope identity or overlap is present and both
antibodies bind to the same or an overlapping epitope on the same
antigen. The anti-idiotype antibody according to the invention is
directed against an antibody specifically binding to the amino acid
sequence of the amyloid .beta. peptide, e.g. in one embodiment to
the amino acid residues of SEQ ID NO: 13.
[0026] The principles of different immunoassays are described, for
example, by Hage, D. S. (Anal. Chem. 71 (1999) 294R-304R). Lu, B.,
et al. (Analyst 121 (1996) 29R-32R) report the orientated
immobilization of antibodies for the use in immunoassays.
Avidin-biotin-mediated immunoassays are reported, for example, by
Wilchek, M., and Bayer, E. A., in Methods Enzymol. 184 (1990)
467-469.
[0027] Monoclonal antibodies and their constant domains contain as
proteins a number of reactive side chains for coupling to a binding
partner, such as a surface, a protein, a polymer (e.g. PEG,
cellulose or polystyrol), an enzyme, or a member of a binding pair.
Chemical reactive groups of antibodies are, for example, amino
groups (lysines, alpha-amino groups), thiol groups (cystines,
cysteines, and methionines), carboxylic acid groups (aspartic
acids, glutamic acids), and sugar-alcoholic groups. Such methods
are e.g. described by Aslam M., and Dent, A., in "Bioconjugation",
MacMillan Ref. Ltd. 1999, pp. 50-100.
[0028] One of the most common reactive groups of proteins is the
aliphatic .epsilon.-amine of the amino acid lysine. In general,
nearly all antibodies contain abundant lysine. Lysine amines are
reasonably good nucleophiles above pH 8.0 (pK.sub.a=9.18) and
therefore react easily and cleanly with a variety of reagents to
form stable bonds. Amine-reactive reagents react primarily with
lysines and the .alpha.-amino groups of proteins. Reactive esters,
particularly N-hydroxy-succinimide (NHS) esters, are among the most
commonly employed reagents for modification of amine groups. The
optimum pH for reaction in an aqueous environment is pH 8.0 to 9.0.
Isothiocyanates are amine-modification reagents and form thiourea
bonds with proteins. They react with protein amines in aqueous
solution (optimally at pH 9.0 to 9.5). Aldehydes react under mild
aqueous conditions with aliphatic and aromatic amines, hydrazines,
and hydrazides to form an imine intermediate (Schiff's base). A
Schiff's base can be selectively reduced with mild or strong
reducing agents (such as sodium borohydride or sodium
cyanoborohydride) to derive a stable alkyl amine bond. Other
reagents that have been used to modify amines are acid anhydrides.
For example, diethylenetriaminepentaacetic anhydride (DTPA) is a
bifunctional chelating agent that contains two amine-reactive
anhydride groups. It can react with N-terminal and .epsilon.-amine
groups of proteins to form amide linkages. The anhydride rings open
to create multivalent, metal-chelating arms able to bind tightly to
metals in a coordination complex.
[0029] Another common reactive group in antibodies is the thiol
residue from the sulfur-containing amino acid cystine and its
reduction product cysteine (or half cystine). Cysteine contains a
free thiol group, which is more nucleophilic than amines and is
generally the most reactive functional group in a protein. Thiols
are generally reactive at neutral pH, and therefore can be coupled
to other molecules selectively in the presence of amines. Since
free sulfhydryl groups are relatively reactive, proteins with these
groups often exist with them in their oxidized form as disulfide
groups or disulfide bonds. In such proteins, reduction of the
disulfide bonds with a reagent such as dithiothreitol (DTT) is
required to generate the reactive free thiol. Thiol-reactive
reagents are those that will couple to thiol groups on proteins,
forming thioether-coupled products. These reagents react rapidly at
slight acidic to neutral pH and therefore can be reacted
selectively in the presence of amine groups. The literature reports
the use of several thiolating crosslinking reagents such as Traut's
reagent (2-iminothiolane), succinimidyl (acetylthio)acetate SATA),
and sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate
(Sulfo-LC-SPDP) to provide efficient ways of introducing multiple
sulfhydryl groups via reactive amino groups. Haloacetyl
derivatives, e.g. iodoacetamides, form thioether bonds and are also
reagents for thiol modification. Further useful reagents are
maleimides. The reaction of maleimides with thiol-reactive reagents
is essentially the same as with iodoacetamides. Maleimides react
rapidly at slight acidic to neutral pH.
[0030] Another common reactive group in antibodies are carboxylic
acids. Proteins contain carboxylic acid groups at the C-terminal
position and within the side chains of aspartic acid and glutamic
acid. The relatively low reactivity of carboxylic acids in water
usually makes it difficult to use these groups to selectively
modify proteins and other biomolecules. When this is done, the
carboxylic acid group is usually converted to a reactive ester by
the use of a water-soluble carbodiimide and reacted with a
nucleophilic reagent such as an amine, hydrazide, or hydrazine. The
amine-containing reagent should be weakly basic in order to react
selectively with the activated carboxylic acid in the presence of
the more highly basic .epsilon.-amines of lysine to form a stable
amide bond. Protein crosslinking can occur when the pH is raised
above 8.0.
[0031] Sodium periodate can be used to oxidize the alcohol part of
a sugar within a carbohydrate moiety attached to an antibody to an
aldehyde. Each aldehyde group can be reacted with an amine,
hydrazide, or hydrazine as described for carboxylic acids. Since
the carbohydrate moiety is predominantly found on the
crystallizable fragment (Fc) region of an antibody, conjugation can
be achieved through site-directed modification of the carbohydrate
away from the antigen-binding site. A Schiffs base intermediate is
formed, which can be reduced to an alkyl amine through the
reduction of the intermediate with sodium cyanoborohydride (mild
and selective) or sodium borohydride (strong) water-soluble
reducing agents.
[0032] The term "sample" includes, but is not limited to, any
quantity of a substance from a living thing or formerly living
thing. Such living things include, but are not limited to, humans,
mice, monkeys, rats, rabbits, and other animals. Such substances
include, but are not limited to, whole blood, serum, or plasma from
an individual, which are the most widely used sources of sample in
clinical routine.
[0033] The term "solid phase" means a non-fluid substance, and
includes particles (including microparticles and beads) made from
materials such as polymer, metal (paramagnetic, ferromagnetic
particles), glass, and ceramic; gel substances such as silica,
alumina, and polymer gels; capillaries, which may be made of
polymer, metal, glass, and/or ceramic; zeolites and other porous
substances; electrodes; microtiter plates; solid strips; and
cuvettes, tubes or other spectrometer sample containers. A solid
phase component of an assay is distinguished from inert solid
surfaces with which the assay may be in contact in that a "solid
phase" contains at least one moiety on its surface, which is
intended to interact with the capture drug antibody. A solid phase
may be a stationary component, such as a tube, strip, cuvette or
microtiter plate, or may be non-stationary components, such as
beads and microparticles. A variety of microparticles that allow
either non-covalent or covalent attachment of proteins and other
substances may be used. Such particles include polymer particles
such as polystyrene and poly (methylmethacrylate); gold particles
such as gold nanoparticles and gold colloids; and ceramic particles
such as silica, glass, and metal oxide particles. See for example
Martin, C. R., et al., Analytical Chemistry-News & Features, 70
(1998) 322A-327A, or Butler, J. E., Methods 22 (2000) 4-23.
[0034] From chromogens (fluorescent or luminescent groups and
dyes), enzymes, NMR-active groups, metal particles, or haptens,
such as digoxygenin, the detectable label is selected in one
embodiment. The detectable label can also be a photoactivatable
crosslinking group, e.g. an azido or an azirine group. Metal
chelates which can be detected by electrochemoluminescence are also
in one embodiment signal-emitting groups, with particular
preference being given to ruthenium chelates, e.g. a ruthenium
(bispyridyl).sub.3.sup.2+ chelate. Suitable ruthenium labeling
groups are described, for example, in EP 0 580 979, WO 90/05301, WO
90/11511, and WO 92/14138.
[0035] The invention provides a method for the immunological
determination of an anti-idiotype antibody against an antibody
against the amyloid .beta. peptide in a sample using an immunoassay
comprising a capture antibody and a tracer antibody. In one
embodiment the immunoassay is an antigen bridging immunoassay. In
another embodiment the immunoassay comprises a capture antibody and
a tracer antibody, wherein the capture antibody is a mixture of the
antibody against the amyloid .beta. peptide comprising at least two
antibodies that differ in the antibody site at which they are
conjugated to the solid phase, and the tracer antibody is a mixture
of the antibody against the amyloid .beta. peptide comprising at
least two antibodies that differ in the antibody site at which they
are conjugated to the detectable label.
[0036] Commonly/generally employed assays have limitations with
respect to the quantification and/or to the determination of an
antibody against the amyloid .beta. peptide in a sample, which
result e.g. from changes in the binding of the antibody against the
amyloid .beta. peptide to the amyloid .beta. peptide. It has been
found that by using an antibody against an antibody against the
amyloid .beta. peptide (anti-anti-A.beta. antibody) according to
the current invention in an immunoassay these limitations can be
overcome.
[0037] The capture antibody useful in a method according to the
invention is conjugated to a solid phase. The conjugation is in one
embodiment performed by chemical binding via N-terminal and/or
.epsilon.-amino groups (lysine), .epsilon.-amino groups of
different lysines, carboxy-, sulfhydryl-, hydroxyl- and/or phenolic
functional groups of the amino acid backbone of the antibody and/or
sugar alcohol groups of the carbohydrate structure of the antibody.
The capture antibody useful in a method according to the invention
is in one embodiment a mixture of at least two antibodies
conjugated to a solid phase, wherein the at least two antibodies
conjugated to a solid phase differ in the site at which they are
conjugated to the solid phase. For example, the mixture of at least
two antibodies conjugated to a solid phase may comprise an antibody
against the amyloid .beta. peptide conjugated via an amino acid of
the amino acid backbone of the antibody to the solid phase and an
antibody against the amyloid .beta. peptide conjugated via a sugar
alcohol group of a carbohydrate structure of the antibody to the
solid phase. Also, for example, the mixture of at least two
antibodies conjugated to a solid phase may comprise antibodies
conjugated to the solid phase via different amino acid residues of
their amino acid backbone. The expression "different amino acid
residue" denotes either two different kinds of amino acids, such as
e.g. lysine and aspartic acid, or tyrosine and glutamic acid, or
two amino acid residues of the amino acid backbone differing in
their position in the amino acid sequence of the antibody. In the
latter case the amino acid can be of the same kind or of different
kind. The expressions "differ in the antibody site" and "site"
denote a difference either in the kind of site, e.g. amino acid or
sugar alcohol group, or in the number of the amino acid of the
amino acid backbone, e.g. at which the antibody is conjugated to
the solid phase. The same applies vice versa to the tracer antibody
useful in a method according to the invention.
[0038] In one embodiment of the invention comprises the heavy chain
variable domain of the antibody against the amyloid .beta. peptide
a CDR3 with an amino acid sequence selected from SEQ ID NO: 1, 2,
or 3. In a further embodiment the light chain variable domain of
said antibody against the amyloid .beta. peptide comprises a CDR3
with an amino acid sequence selected from SEQ ID NO: 4, 5, or 6. In
a further embodiment the antibody against the amyloid .beta.
peptide comprises a heavy chain variable domain with an amino acid
sequence selected from SEQ ID NO: 7, 8, or 9. In still a further
embodiment said antibody against the amyloid .beta. peptide
comprises a light chain variable domain with an amino acid sequence
selected from SEQ ID NO: 10, 11, or 12.
[0039] In one embodiment of the method according to the invention
the immunoassay comprises as capture antibody the F(ab').sub.2
fragment of a monoclonal antibody against the amyloid .beta.
peptide, which is biotinylated and conjugated via streptavidin to a
solid phase, and as tracer antibody a polyclonal antibody against
human immunoglobulin G conjugated to horseradish peroxidase.
[0040] In another embodiment of the method according to the
invention the immunoassay is a double antigen bridging
immunoassay.
[0041] In a further embodiment of the method according to the
invention the immunoassay comprises a capture antibody, a tracer
antibody and a detection antibody, wherein the capture antibody is
a biotinylated antibody against the amyloid .beta. peptide
conjugated to a solid phase via streptavidin, the tracer antibody
is an antibody against the amyloid .beta. peptide conjugated to
digoxygenin, and the detection antibody is an antibody against
digoxygenin conjugated to horseradish peroxidase.
[0042] In another embodiment of the method according to the
invention the immunoassay comprises a capture antibody, a tracer
antibody and a detection antibody, wherein the capture antibody is
a biotinylated anti-idiotype antibody against an antibody against
the amyloid .beta. peptide conjugated to a solid phase via
streptavidin, the tracer antibody is an anti-idiotype antibody
against an antibody against the amyloid .beta. peptide conjugated
to digoxygenin as detectable label, and the detection antibody is
an antibody against digoxygenin conjugated to horseradish
peroxidase.
[0043] In another embodiment of the method according to the
invention the immunoassay comprises the amyloid .beta. peptide
conjugated to a solid phase, an anti-idiotype antibody against an
antibody against the amyloid .beta. peptide conjugated to
digoxygenin as detectable label, and a detection antibody against
digoxygenin conjugated to horseradish peroxidase.
[0044] The mouse-mouse hybridoma cell line according to the
invention, hybridoma cell line MAK<Mab31>M-1.5.74, was
deposited under the Budapest Treaty on the international
recognition of the deposit of microorganisms for the purposes of
patent procedure, with Deutsche Sammlung von Mikroorganismen and
Zellkulturen GmbH (DSMZ), Germany, on Jul. 29, 2008 under Accession
No. DSM ACC2939.
[0045] The following examples, sequence listing and figures are
provided to aid the understanding of the present invention, the
true scope of which is set forth in the appended claims. It is
understood that modifications can be made in the procedures set
forth without departing from the spirit of the invention.
DESCRIPTION OF THE FIGURES
[0046] FIG. 1 Selection of anti-idiotype anti-human A.beta.
antibody.
[0047] FIG. 2 Assay to evaluate cross-reactivity.
[0048] FIG. 3 Quantification of anti-human A.beta. antibody.
[0049] FIG. 4 Alternative assay for quantification of anti-human
A.beta. antibody.
[0050] FIG. 5 Detection of anti-anti-human A.beta. antibody
antibodies (ADA).
[0051] FIG. 6 Detection of neutralizing anti-anti-human A.beta.
antibody antibodies.
EXAMPLES
Example 1
Preparation of the F(ab').sub.2 Fragment of Antibody Against the
Amyloid .beta. Peptide
[0052] For example, an exemplary antibody against the amyloid
.beta. peptide (anti-A.beta. antibody) and its corresponding
nucleic acid sequences are reported in WO 2003/070760 or US
2005/0169925 or in SEQ ID NO: 1 to 12.
[0053] The anti-A.beta. antibody in 100 mM sodium citrate buffer,
pH 3.6, was incubated with pepsin (5 .mu.g pepsin per mg antibody).
The fragmentation was analyzed by analytical gel filtration and
stopped after 50 minutes by adjusting the pH to 7.0 with potassium
phosphate. After dialysis of the mixture against 50 mM potassium
phosphate buffer containing 300 mM sodium chloride at pH 7.5 the
preparation was concentrated to about 20 mg/ml and applied to a gel
filtration column (Superdex 200). The recovered fractions were
analyzed by analytical gel filtration and the fractions containing
the F(ab').sub.2 fragment were applied to an affinity matrix with
immobilized polyclonal antibodies against human Fc.gamma. to
eliminate trace amounts of Fc.gamma. containing fragments. The flow
through was pooled and finally concentrated to about 20 mg/ml.
Example 2
Generation of Monoclonal Anti-Idiotypic Antibodies
[0054] a) Immunization of mice
[0055] Female Balb/c or NMRI mice, respectively, 8-12 weeks of age,
were primarily immunized intraperitoneally with 100 .mu.g of the
F(ab').sub.2 fragment of the monoclonal anti-A.beta. antibody
comprising a heavy chain variable domain selected from SEQ ID NO: 7
to 9 and a light chain variable domain selected from SEQ ID NO: 10
to 12 mixed with CFA (Complete Freund's Adjuvant) obtained as
outlined in Example 1. Three further intraperitoneal immunization
steps followed after 4, 7, and 10 weeks, with application of 100
.mu.g of the above-mentioned F(ab').sub.2 fragment per mouse mixed
with IFA (Incomplete Freund's Adjuvant). Subsequently, intravenous
or intraperitoneal, respectively, boost immunizations were done,
each with 25 to 100 .mu.g of F(ab').sub.2 fragment in PBS
(phosphate buffered saline) three days before the fusion.
b) Fusion and Cloning
[0056] Fusion with myeloma cells derived from the spleen cells of
the mice immunized according to a) was performed according to
Galfree, G., and Milstein, C. (Galfre, G., and Milstein, C.,
Methods Enzymol. 73 (1981) 3-46). Approximately 1.times.10.sup.8
splenocytes were mixed with 2.times.10.sup.7 myeloma cells
(P3.times.63-Ag8.653, ATCC CRL1580) and centrifuged (10 min. at
300.times.g at 4.degree. C.). The cells were washed afterwards once
with the culture medium RPMI 1640 without FCS (fetal calf serum),
and centrifuged again at 400.times.g in a 50 ml pointed vial.
Thereafter, one ml of PEG (poly (ethylene glycol), molecular weight
4,000 g/mol) was added. Mixing was done by the pipetting. After 1
min. in a water bath at 37.degree. C., 5 ml of RPMI 1640 without
FCS were added drop wise. The suspension was mixed, filled up to 50
ml with RPMI 1640 containing 10% (v/v) FCS, and centrifuged
afterwards. The sedimented cells were resuspended in RPMI 1640 with
10% FCS, and plated in hypoxanthine-azaserine selection medium (100
mmol/l hypoxanthine, 1 .mu.g/ml azaserine in RPMI 1640 with 10%
FCS) containing the growth factor Interleukin 6 (IL-6, 100 U/ml).
After approximately 10 days, the primary cultures were assayed for
specific antibody synthesis (see Example 3). Primary cultures
exhibiting binding to the above-mentioned F(ab').sub.2 fragment as
well as no cross reaction with Human IgG, and having been shown to
be anti-idiotypic were individualized by single cell deposition
into 96-well cell culture plates using a flow cytometer (FACSAria,
BD Biosciences), with the medium containing the growth factor
Interleukin 6 (100 U/ml). By following this protocol, the deposited
clone 1.5.74 was generated (Table 1). The cell line useful in the
current invention was deposited with the Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSMZ), Germany.
TABLE-US-00001 TABLE 1 Clone producing anti-idiotypic monoclonal
antibody IgG Class and Clone Subclass Deposition No. Date of
deposit 1.5.74 IgGX, kappa DSM ACC2939 29.07.2008
c) Production of Immunoglobulin from the Cell Culture
Supernatant
[0057] The generated hybridoma cell line was inoculated at an
initial cell density of 1.0.times.10.sup.5 cells per ml in RPMI
1640 medium supplemented with 10% FCS, and expanded in a spinner
culture. From there, a Miniperm unit was inoculated with the cells
at an initial cell density (live cells) of 2.4.times.10.sup.6 cells
per ml in RPMI 1640 medium supplemented with 10% FCS, and expanded.
In the harvested culture supernatant, a concentration of 1.2 mg of
monoclonal antibody per ml was achieved. Purification of the
antibody from the culture supernatant was done according to
standard protein chemical methods, e.g. according to Bruck, C., et
al., Methods Enzymol. 121 (1986) 587-596.
Example 3
Screening Assays for Detection of Anti-Idiotypic Antibodies
a) Primary Screening for Antibodies Binding to the Drug
Antibody
[0058] For the determination of the specificity of the antibodies
in the culture supernatants of the hybridoma cells, MTPs
(microtiter plates) pre-coated with recombinant streptavidin
(MicroCoat, Bernried, Germany) were coated with biotinylated
monoclonal anti-A.beta. antibody, 200 ng/ml, or biotinylated human
IgG, 2 .mu.g/ml, respectively, in PBS supplemented with 1.0% (w/v)
BSA fraction II (100 .mu.l per well, 60 min. incubation at ambient
temperature, with shaking) Subsequently the wells were washed three
times with 0.9% (w/v) sodium chloride solution containing 0.05%
(w/v) Tween.RTM. 20. In the next step, per well 100 .mu.l of the
antibody solution to be assayed (culture supernatant) were added,
and incubated for 60 min. at ambient temperature, with shaking.
After three wash steps with 0.9.degree. A (w/v) NaCl/0.05.degree. A
Tween.RTM. 20 per well, 100 .mu.l of a horseradish
peroxidase-labeled F(ab').sub.2 fragment of a polyclonal sheep
anti-mouse Fc.gamma. antibody were added and incubated for 60 min.
at ambient temperature, with shaking Subsequently, washing was
performed as above. Finally, per well 100 .mu.l of ABTS.RTM. (Roche
Diagnostics GmbH, Germany, catalog no. 1684302) were added. After
30 min. incubation at ambient temperature, the extinction (optical
density OD) was measured at 405 and 492 nm [405/492] in a
commercial microtiter plate ELISA reader. This screening led to a
selection of antibodies binding well to monoclonal anti-A.beta.
antibody IgG as well as exhibiting only a low or even no cross
reactivity to human IgG. This selection of antibodies was further
subjected to assay b).
b) Selection of Anti-Idiotypic Antibodies
[0059] In order to identify, from the selection of antibodies of
the primary screening a), those that are anti-idiotypic, the assay
described in the following was performed. MTPs pre-coated with
recombinant streptavidin (MicroCoat, Bernried, Germany) were coated
with biotinylated A.beta. peptide, 250 ng/ml, in PBS with 0.5% BSA
fraction II (100 .mu.l per well, 60 min. incubation at ambient
temperature, with shaking), and subsequently washed three times
with 0.9% (w/v) NaCl/0.05% Tween.RTM. 20. In the next step, per
well 50 .mu.l of the digoxygenin-labeled Fab fragment of monoclonal
anti-A.beta. antibody, at 2.5-15 ng/ml, and 50 .mu.l of PBS
(reference signal), or 50 .mu.l of the candidate antibodies
(culture supernatant; assay signal), respectively, were added, and
incubated for 60 min. at ambient temperature, with shaking. After
three wash steps with 0.9% (w/v) NaCl/0.05% Tween.RTM. 20 per well,
100 .mu.l of a horseradish peroxidase-labeled Fab fragment of a
polyclonal sheep anti-digoxygenin antibody were added for the
detection of bound human monoclonal anti-A.beta.
antibody-digoxygenin conjugate, and incubated for 60 min. at
ambient temperature, with shaking. Subsequently, washing was
performed as above. Finally, per well 100 .mu.l of ABTS.RTM. (Roche
Diagnostics GmbH, Mannheim, Germany, catalog no. 1684302) were
added. After 30 min. incubation at ambient temperature, the
extinction was measured at [405/492] nm in a commercial microtiter
plate ELISA reader. Antibodies exhibiting a strongly reduced assay
signal, as compared to the associated reference signal, were
selected as candidates for further use.
c) Selection of Antibodies with the Lowest Cross Reactivity to
Human IgG
[0060] In order to identify, from the candidates of screening b),
those that exhibit the lowest cross reactivity to human IgG, the
assay described in the following was performed. MTPs pre-coated
with recombinant streptavidin (MicroCoat, Bernried, Germany) were
coated with biotinylated monoclonal anti-A.beta. antibody IgG, 50
ng/ml, in a buffer (125 .mu.l per well, 30 min. incubation at
ambient temperature, with shaking), and subsequently washed three
times with 0.9% (w/v) NaCl/0.05% Tween.RTM. 20. In the next step,
per well 100 .mu.l of a mix of 100 .mu.l of the respective
candidate antibody (culture supernatant), 100 .mu.l of human IgG
(at concentrations up to 73 mg/ml), and 200 .mu.l of
digoxygenin-labeled monoclonal anti-A.beta. antibody-Fab fragment,
were added, and incubated for 60 min. at ambient temperature, with
shaking. After three wash steps with 0.9% (w/v) NaCl/0.05%
Tween.RTM. 20 per well, 100 .mu.l of a horseradish
peroxidase-labeled Fab fragment of a polyclonal sheep
anti-digoxygenin antibody were added for the detection of bound
monoclonal anti-A.beta. antibody-Fab-digoxygenin conjugate, and
incubated for 60 min. at ambient temperature, with shaking.
Subsequently, washing was performed as above. Finally, per well 100
.mu.l of ABTS.RTM. (Roche Diagnostics GmbH, Mannheim, Germany,
catalog no. 1684302) were added. After 30 min. incubation at
ambient temperature, the extinction was measured at [405/492] nm in
a commercial microtiter plate ELISA reader. Antibodies exhibiting
the least loss of assay signal by the addition of human IgG, as
compared to without addition of human IgG, were selected for
further use.
Example 4
Purification of Mouse Monoclonal Anti-Idiotypic Antibodies Directed
Against Monoclonal Anti-A.beta. Antibody
[0061] The fermentation supernatant of the antibody against
monoclonal anti-A.beta. antibody IgG was concentrated about tenfold
and transferred to a buffer with 20 mM Tris, 1 M ammonium sulfate,
pH 9.0, and applied to protein A-sepharose. The eluate with 0.2 M
sodium citrate and 0.2 M ammonium sulfate at pH 5.5 was dialyzed
against phosphate buffer, pH 7.5. Contaminants of bovine IgG (from
FCS in the fermentation broth) were separated by immunoadsorption
with immobilized antibodies against bovine IgG.
Example 5
Preparation of Biotinylated Antibody Against Monoclonal
Anti-A.beta. Antibody
[0062] The antibody against monoclonal anti-A.beta. antibody-IgG in
phosphate buffer, pH 8.5, was adjusted to a protein concentration
of about 15 mg/ml. D-biotinoyl-aminocaproic
acid-N-hydroxysuccinimide was dissolved in DMSO and added to the
solution in a molar ratio of 1:5. The reaction was stopped after 60
minutes by adding L-lysine, and the surplus of the labeling reagent
was removed by dialysis against 25 mM potassium phosphate buffer,
with 150 mM sodium chloride, pH 7.5, and by gel filtration.
Example 6
Preparation of Digoxigenylated Antibody Against Monoclonal
Anti-A.beta. Antibody
[0063] The antibody against monoclonal anti-A.beta. antibody-IgG in
phosphate buffer, pH 8.5, was adjusted to a protein concentration
of about 15 mg/ml. Digoxygenin
3-O-methylcarbonyl-.epsilon.-aminocaproic acid-N-hydroxysuccinimide
was dissolved in DMSO and added to the antibody solution in a molar
ratio of 1:5. The reaction was stopped after 60 minutes by adding
L-lysine, and the surplus of the labeling reagent was removed by
dialysis against 25 mM potassium phosphate buffer, with 150 mM
sodium chloride, pH 7.5, and by gel filtration.
Example 7
Assay for Quantification of Monoclonal Anti-A.beta. Antibody
[0064] The ELISA is an assay for the quantitative determination of
monoclonal anti-A.beta. antibody in human plasma using streptavidin
micro titer plates (MTP). The calibration standards and the samples
are pre-incubated with a biotinylated and digoxigenylated
anti-idiotypic antibody directed against the idiotype of the
monoclonal anti-A.beta. antibody (-IgG-Bi and -IgG-Dig). After 30
min. pre-incubation the mixture is transferred to the micro titer
plate. The Sandwich-ELISA is detected with sheep anti-digoxygenin
antibody conjugated to a peroxidase which catalyzes the color
reaction of the ABTS.RTM. substrate. The signal is determined by an
ELISA reader. Washing steps are done before and after incubation
with anti-DIG pAb-Fab-POD (pAb=polyclonal antibody). All
calibration standards and samples include 10% human plasma.
Example 8
Assay for Detection of an Anti-Drug Antibody Directed Against
Monoclonal Anti-A.beta. Antibody
[0065] Biotinylated monoclonal anti-A.beta. antibody has been
conjugated to (bound onto) the wells of a streptavidin-coated
microtiterplate (SA-MTP) in the first step. Not conjugated
(unbound) antibody was removed by washing with universal buffer.
Afterwards the samples and the reference standards (monoclonal
anti-idiotypic anti-A.beta. antibody spiked in 5% human serum) have
been incubated in the wells. Anti anti-A.beta. antibody bound to
the immobilized monoclonal anti-A.beta. antibody. After having
washed away unbound substances the bound anti anti-A.beta. antibody
was detected with digoxigenylated monoclonal anti-A.beta. antibody
followed by incubation with a horse-radish peroxidase labeled
anti-digoxygenin-antibody (see FIG. 1). The antibody-enzyme
conjugate catalyzed the color reaction of the ABTS.RTM. substrate.
The signal was measured by ELISA reader at 405 nm (reference
wavelength: 490 nm). Absorbance values of each serum sample were
determined in triplicate.
Example 9
Alternative Assay for Quantification of Monoclonal Anti-A.beta.
Antibody
[0066] The ELISA is an assay for the quantitative determination of
monoclonal anti-A.beta. antibody in human plasma using micro titer
plates (MTP) coated with amyloid beta protein comprising amino
acids 1-40 (A.beta.(1-40)). The calibrations standards and samples
are incubated in the wells of micro titer plates coated with
A.beta.(1-40) and the amount of monoclonal anti-A.beta. antibody
bound to A.beta.(1-40) coated surface is detected with
digoxigenylated anti-idiotypic antibody directed against the
idiotype of monoclonal anti-A.beta. antibody (-IgG-Dig) and
anti-digoxygenin antibody conjugated to peroxidase which catalyzes
the color reaction of the ABTS.RTM. substrate. The signal is
measured by an ELISA reader.
[0067] Washing steps are done between all incubation cycles. After
coating of the micro titer plate (MTP) with A.beta.(1-40) an
additional blocking step is necessary (with incubation buffer). All
calibration standards and samples include 10% human plasma. All
incubation steps are done at room temperature.
Example 10
Assay for Detection of Neutralizing Anti-Drug Antibodies Directed
Against Monoclonal Anti-A.beta. Antibody
[0068] For analysis of neutralizing anti-drug antibodies directed
against the complementarity-determining region of the monoclonal
therapeutic anti-A.beta. antibody, a competition ELISA was
developed. Plasma samples or anti-idiotypic antibodies against
monoclonal anti-A.beta. antibody as standard are pre-incubated with
monoclonal anti-A.beta. antibody-DIG conjugate. Remaining free
monoclonal anti-A.beta. antibody-DIG conjugate is captured on an
A.beta.-coated microtiter plate and detected with a
peroxidase-labeled anti-digoxygenin antibody and subsequent color
reaction with ABTS.
Sequence CWU 1
1
13113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr Phe Asp
Val1 5 10217PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 2Gly Lys Gly Asn Thr His Lys Pro Tyr Gly
Tyr Val Arg Tyr Phe Asp1 5 10 15Val315PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3Leu
Leu Ser Arg Gly Tyr Asn Gly Tyr Tyr His Lys Phe Asp Val1 5 10
1548PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Gln Gln Val Tyr Asn Pro Pro Val1
558PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Phe Gln Leu Tyr Ser Asp Pro Phe1
568PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Gln Gln Leu Ser Ser Phe Pro Pro1
57122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Thr
His Tyr Ala Arg Tyr Tyr Arg Tyr Phe Asp Val Trp 100 105 110Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 1208126PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Lys Gly Asn Thr His Lys
Pro Tyr Gly Tyr Val Arg Tyr 100 105 110Phe Asp Val Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 1259124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
9Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Leu Ser Arg Gly Tyr Asn
Gly Tyr Tyr His Lys Phe Asp 100 105 110Val Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 12010110PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 10Asp Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Val Tyr Asn Pro Pro
85 90 95Val Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 11011110PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 11Asp Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser
Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55 60Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu65 70 75 80Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Leu Tyr Ser Asp Pro 85 90 95Phe
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105
11012110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Val Pro Ala Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu65 70 75 80Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Leu Ser Ser Phe Pro 85 90 95Pro Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 1101342PRTHomo
sapiens 13Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His
Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly
Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala 35
40
* * * * *